1) Field of the Invention
This invention relates to a technique for introducing a wild-type DHFR gene, which codes for dihydrofolate reductase, as a dominant selective marker into wild-type animal cells to obtain a transformant and then culturing the transformant in an amplification medium containing a DHFR inhibitor to obtain an amplified transformant having amplified productivity of a useful substance. This invention can be used effectively as a technique for the production of tissue plasminogen activator (hereinafter abbreviated as "t-PA").
An object of this invention is to obtain an amplified transformant from a transformant, which itself contains a desired gene, by a specific method. It is another feature of this invention that the amplified transformant contains a foreign wild-type DHFR gene.
2. Description of the Prior Art
A DHFR gene codes for dihydrofolate reductase, which takes part in the biosynthesis of DNA and RNA, and the gene can be amplified within cells resistant to methotrexate (MTX) which is known as a carcinostatic agent.
Attempts have been made to amplify a desired foreign gene within a transformant by use of DHFR gene amplification so that the production level of a foreign gene product could be improved.
Co-amplification of the DHFR gene and the desired foreign gene is performed by culturing a transformant, which contains the DHFR gene and the foreign gene, in a fresh medium with a higher concentration of MTX and screening a strain resistant to the higher MTX concentration by using the DHFR gene as a marker. This operation may be repeated in a much higher concentration of MTX.
As useful DHFR gene sources, there are both wild-type and mutant-type. When a wild-type DHFR gene is chosen, a DHFR-deficient strain is generally used as a host.
As one example of the application of a mutant DHFR gene, European Patent Publication No. 117059-A discloses a technique for obtaining t-PA producing transformants by using a mutant DHFR gene as a dominant selective marker gene for wild-type cells to give MTX (methotrexate) resistance to the cells, selecting the transformant which was co-transfected with a human t-PA gene and then culturing the thus-obtained transformant in the presence of MTX at higher concentration so as to increase both of the DHFR and t-PA genes.
The above conventional technique is however accompanied by problems. Firstly, the mutant DHFR gene yields due to its mutation a genetic product in which the 22th amino acid has been changed from leucine to arginine. Possible adverse effects such as malignant growth which such a mutant DHFR gene or its genetic product may cause on the wild-type cells employed as a host, have not been fully investigated. As a pharmaceutical technique which is supposed to meet stringent safety requirements, the use of the mutant DHFR gene is still considered to involve unsolved problems.
The mutant DHFR gene is different in its binding affinity to MTX from wild-type DHFR genes and the binding affinity of the former gene is as weak as one two hundred seventieth of the binding affinity of the latter.
In a series of gene multiplication operations for obtaining an MTX-resistant strain in the presence of MTX at an increased concentration, it is accordingly indispensable to culture transformed cells with the mutant DHFR gene at a considerably higher MTX concentration when compared to the case of using wild-type DHFR genes.
The MTX concentration required for the culture of the amplified transformants is much higher than that employed when wild-type DHFR genes are used. Since such a high MTX concentration is harmful to cells, a resistant strain obtained in the presence of MTX at such a high concentration is itself unstable. Under the current technology, the above described selection method in which a high MTX concentration was employed to obtain amplified transformants has not yet resulted in any reproducible and practical technique.
For the reasons mentioned above, the method for forming a high t-PA producing transformant by use of a mutant DHFR gene is still believed to be incomplete as a technique to be used for the production of pharmaceutical products.
Wild-type cells capable of expressing their DHFR gene are usually chosen as host animal cells, because mutant cells are not easily established in the case of animal cells and therefore the mutant cells are not advantageous for industrial production. However, a transformant may not always be obtained stably when a wild-type DHFR gene is used as a dominant selective marker gene for such wild-type cells. Even if a transformant could be obtained, the expression of the characters by the transformant is unexceptionally unstable. Difficulties are therefore encountered in many instances upon screening a transformant by this method. As a matter of fact, no stable t-PA producing strain was obtained when a wild-type DHFR gene was used for wild-type Chinese hamster cells (European Patent Publication No. 117060-A).
It may therefore be contemplated that a wild-type DHFR gene could be used as a selective marker gene if a DHFR-deficient strain can be established from wild-type cells. This approach is however not easy, because the establishment of such a DHFR-deficient strain per se is complex and time-consuming. [For details, reference may be had to Urlanb, G., & Chasin, L. A., Proc. Natl. Acad. Sci. U.S.A., 77, 4216 (1980).]